Cardiovascular complications are the leading cause of death in patients in diabetic patients [21, 22]. The current study provides evidence to support that excess accumulation of advanced glycation end products (AGEs) and inflammation is emerging as important mechanism for micro- and macrovascular complication of diabetes [23, 24]. Monocytes transmigrate into the subendothelial space and differentiate into macrophages, which migrate, infiltrate and accumulate in the vascular tissues, involving in diabetic vascular complications. Clinical study has also observed significant increase of AGEs accumulation in diabetic vascular tissues , which may induce macrophage migration across an endothelial cell monolayer .
Heparanase (HPA), a mammalian endo-beta-D-glu-curoxnidase, has previously been shown to be a key enzyme in the metastatic potential of tumor-derived cells and cells of the immune system [27–29]. Recently, evidences show that HPA enhances the phosphorylation of selected signaling molecules, in a manner that is mediated by its C-terminal domain but not enzymatic activity [12, 30]. In the study, we have characterized the role and mechanism of HPA in AGEs-induced macrophage migration independent of enzymatic activity.
With the MTT assay we observed the dose-dependent reduction of viability in macrophages treated by AGEs. The viability revealed a nonlinear dose response to AGEs in macrophages. AGEs treatment at 25, 50 and 100 mg/L for 3,6,12 and 24 h incubation didn’t result in a significant loss of viability in macrophages compared with controls (0 mg/L) (Figure 1A). We then used 100 mg/L AGEs, a max dosage which didn’t changed the viability significantly within studied AGEs concentration range at 24 h culture, for further studies on the role of HPA in macrophages.
Moreover, pretreatment with LY294002 (7.5-15 μM), anti-RAGE or HPA antibody (10 μg/ml) for 1 h before culture with 100 mg/L AGEs for 3,6,12 and 24 h incubation didn’t result in a significant loss of viability in macrophages compared with controls (Figure 1B). We then used preincubation of 15 μM LY294002, 10 μg/ml anti-RAGE or HPA antibody, the dosage which didn’t changed the viability significantly within the studied concentration range at 24 h culture, for further studies in 100 mg/L AGEs-induced macrophages.
Evidences have shown that HPA play a role in signaling pathway . C-terminus domain mediates nonenzymatic functions of HPA, facilitating the phosphorylation of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), in an enzymatic activity-independent manner in pituitary tumor and proximal tubular [32, 33]. Recent studies show that AKT (protein kinase B), a serine/threonine protein kinase, regulates monocyte/macrophage migration. Inhibition of AKT pathways decreased macrophage migration while mammalian cell migration can be promoted by enhancing AKT signaling [34–36].
In agreement with previous study [26, 37], we have shown that AGEs induced macrophage migration significantly. However, more importantly, we have shown that pretreatment of anti-HPA antibody, which bind to the C-terminus domain of HPA specially, inhibited the macrophage migration significantly (Figure 2). This demonstrates that C-terminus domain of HPA mediates AGEs-induced macrophage migration. We speculate that the C-terminus domain of HPA may mediate AGEs-induced macrophage migration via AKT signaling pathway.
Subsequently, we detected the role of PI3K/AKT signaling pathway in AGEs-induced macrophage migration via HPA. AKT is a target of PI3K activation and its phosphorylation is prevented by PI3K/AKT inhibitors. Although AKT phosphorylation is increased by external HPA in a short time, the AKT phosphorylation induced by changes of HPA protein after AGEs treatment may need longer time. So we chose to determine the levels of AKT at 24 h culture. The results show that the levels of AKT phosphorylation was increased at 24 h in macrophage cultured with AGEs. Pretreatment with an antibody recognizing the C-terminus domain of HPA protein inhibited AKT phosphorylation significantly (Figure 4). The results suggest that AGEs could activate PI3K/AKT signaling pathway via C-terminus domain of HPA protein. Furthermore, we observed that AGEs-induced cell migration was attenuated by using LY294002, a PI3K/AKT inhibitor, in macrophages (Figure 2). These data indicate that the AGEs-induced macrophage migration is partially mediated by the activation of PI3K/AKT signaling pathway in HPA-dependent manner. HPA-PI3K/AKT signal pathway may be important in macrophage migration induced by AGEs.
Recently, it was reported that AGEs could induce HPA expression through receptor for advanced glycation end products (RAGE) in human microvascular endothelial cells . Substantial evidence has demonstrated that RAGE plays a central role in the etiology of diabetes complications and inflammation [38–40]. RAGE ligand, such as HMGB-1, could stimulate phosphorylation of AKT and cell proliferation/migration through RAGE/PI3K/AKT signal transduction pathway [41, 42]. Here we assess the role of RAGE on HPA expression and AKT pathway which associated with macrophage migration.
We observed that treatment with AGEs for 24 h culture resulted into significant increase of HPA mRNA, HPA latent form (65 kDa) and enzyme form (50 kDa) compared with untreated cells. 50 kDa enzyme form is more strongly induced than 65 kDa latent form. All the changes could be attenuated by pretreatment with anti-RAGE antibody (Figure 3A and 3C). These results show that increased expression of HPA mRNA and protein are associated with AGEs stimulation via RAGE and HPA protein expression may partly dependent of post-transcriptional regulation in macrophages.
Furthermore, ELISA analysis was employed to determine the levels of HPA in supernatant. We discovered that pretreatment with anti-RAGE antibody inhibited the increased secretion of HPA in AGEs-stimulated macrophages significantly (Figure 3B). The result from ELISA suggests that AGEs could induce HPA protein secretion via RAGE as well as the results from Western blot. These data provide the first evidence for AGEs-induced macrophage HPA mRNA, protein expression and secretion in a RAGE-dependent manner. RAGE-HPA pathway may play a key role in AGEs-induce macrophage migration.
To analyze the relation between RAGE, AKT phosphorylation and migration to further demonstrate RAGE-HPA-PI3K/AKT pathway, an antibody against RAGE is used to block the effect of RAGE in AGEs-induced macrophages. The results show that pretreatment of the cells with the blocking antibody to RAGE suppressed AGEs-induced AKT phosphorylation (Figure 4) and cell migration significantly in macrophages (Figure 2). The result suggests that RAGE could mediate AGEs-induced macrophage migration via AKT pathway and agrees to RAGE-HPA-PI3K/AKT pathway associated with AGEs-stimulated macrophage migration.